Helicobacter infections of human gastric epithelium cause gastritis, are a major factor in the development of peptic ulcers and gastric lymphoma, and may be a risk factor for the development of gastric cancer. (Blaser, M. J. "Gastric Campylobacter-like Organisms, Gastritis and Peptic Ulcer Disease" Gastroenterology, vol. 93, 371-383 (1987); Graham, D. Y. "Campylobacter pylori and Peptic Ulcer Disease" Gastroenterology, vol. 196, 615-625 (1989); Parsonnet, J. et al. "Helicobacter pylori Infection in Intestinal and Diffuse-Type Gastric Adenocarcinomas" J. Natl. Cancer Inst., vol. 93, 640-643 (1991); Wotherspoon, A. C., et al., "Regression of Primary Low-Grade B-Cell Gastric Lymphoma of Mucosa-Associated Lymphoid Tissue Type After Eradication of Helicobacter pylori," Lancet, vol. 342, 575-577 (1993)). The most frequent infection agent is Helicobacter pylori, followed at a much lower frequency by Helicobacter heilmanii. Helicobacter pylori is a slender S-shaped gram negative microorganism, which is routinely recovered from gastric biopsies of adults and children with histologic evidence of gastritis or peptic ulceration. Evidence for a causal elationship between Helicobacter pylori and gastroduodenal disease comes from studies in human volunteers, patients with ulcers and gastric cancer, gnotobiotic pigs, and germ-free rodents. Regarding etiology, Koch's postulates were satisfied by creating histologically confirmed gastritis in previously uninfected individuals following consumption of viable microorganisms. (Marshall, B. J. et al. "Attempt to Fulfill Koch's Postulate for pyloric Campylobacter" Med. J. Aust., vol. 142, 436-439 (1985); Morris, A. et al. "Ingestion of Campylobacter pyloritis Causes Gastritis and Raised Fasting Gastric pH" Am. J. Gastroenterol., vol. 82, 192-199 (1987); Engstrand, L. et al. "Inoculation of Barrier-Born Pigs With Helicobacter pylori: A Useful Animal Model for Gastritis Type B" Infect. Immun., vol. 53, 1763-1768 (1990); Fox, J. G. et al. "Gastric Colonization by Campylobacter pylori Subsp. mustelae in Ferrets" Infect. Immun., vol. 56, 2994-2996 (1988); Fox, J. G. et al. "Helicobacter mustelae-Associated Gastritis in Ferrets: An Animal Model of Helicobacter pylori Gastritis in Humans" Gastroenterology, vol. 99, 352-361 (1990); Lee, A. et al. "A Small Animal Model of Human Helicobacter pylori Active Chronic Gastritis" Gastroenterology, vol. 99, 1315-1323 (1990); Fox, J. G. et al. "Helicobacter Felis Gastritis in Gnotobiotic Rats: An Animal Model of Helicobacter pylori Gastritis" Infect. Immun., vol. 59, 785-791 (1991); Eaton, K. A. et al. "Campylobacter pylori Virulence Factors in Gnotobiotic Piglets" Infect. Immun., vol. 57, 1119-1125 (1989)), and by treatment to eradicate Helicobacter pylori, with resolution of the gastritis and, in patients with peptic ulcer disease, a decrease in the recurrence rate. (Peterson, W. L. "Helicobacter pylori and Peptic Ulcer Disease" N. Engl. J. Med., vol. 324, 1043-1048 (1991)).
Gastroduodenal diseases thought to be associated with Helicobacter infection include acute, chronic, and atrophic gastritis, peptic ulcer disease including both gastric and duodenal ulcers, gastric cancer, chronic dyspepsia with severe erosive gastroduodenitis, refractory non-ulcer dyspepsia, intestinal metaplasia, and low grade MALT lymphoma. Helicobacter infection is also the principle cause of asymptomatic chronic gastritis.
In spite of in vitro susceptibility to many antimicrobial agents, in vivo eradication of established Helicobacter pylori infections with antimicrobial agents is often difficult to achieve. (Czinn, S. J. and Nedrud, J. G. "Oral Immunization Against Helicobacter pylori" Infect. Immun., vol. 59, 2359-2363 (1991)). The microorganism is found within the mucous coat overlying the gastric epithelium and in gastric pits. These are locations which do not appear to allow for adequate antimicrobial levels to be achieved even when antibiotics are given orally at high doses. At the present time, most authorities recommend a "triple therapy", namely a bismuth salt in combination with drugs such as tetracycline and metronidazole for 2-4 weeks. However, the effectiveness of this or other chemotherapeutic regimens remains suboptimal. Recently, a National Institutes of Health panel of medical experts recommended a triple therapy with bismuth, tetracycline and metronidazole, administered for two weeks for treatment of peptic ulcers (Cimons, M., "Drug Combination Found Effective on Peptic Ulcers," L.A. Times at A14 (Feb. 10, 1994)). However, this treatment is commonly associated with diarrhea and it may produce serious adverse drug reactions. (See, Dick-Hegedus, E. and Lee, A., "Use of a Mouse Model to Examine Anti-Helicobacter pylori Agents," Scand. J. Gastroenterol., vol. 26, 909-915 (1991)). Treatment with antibiotics also may not solve the problem of reinfection and there is evidence for a high incidence of reinfection in some studies (Coelho, L. G., et al., "Duodenal Ulcer and Eradication of H. pylori in a Developing Country: An 18-Month Follow-Up Study," Scand. J. Gastroenterol. vol. 27, 362-66 (1992)). Therefore there is a great need for a vaccine that can be used to treat infection and to prevent future infections.
At the present time little is known regarding the role of the mucosal immune systems in the stomach. The distribution of immunoglobulin (Ig) producing cells in the normal gastric antrum indicates that IgA plasma cells make up 80% of the total plasma cell population. In addition, the number of plasma IgA cells present in the gastric antrum is comparable to other mucus membranes. (Brandtzaeg, P. "Role of J Chain and Secretory Component in Receptor-Mediated Glandular and Hepatic Transport of Immunoglobulins in Man" Scand. J. Immunol., vol. 22, 111-146 (1985); Brandtzaeg, P. et al. "Production and Secretion of Immunoglobulins in the Gastrointestinal Tract" Ann. Allergy, vol. 59, 21-39 (November, 1987)). A number of studies in humans (Wyatt, J. I. et al. "Local Immune Response to Gastritis Campylobacter in Non-ulcer Dyspepsia" J. Clin. Path., vol. 39, 863-870 (1986)), and in animal models (Fox, J. G. et al. "Helicobacter mustelae-Associated Gastritis in Ferrets: An Animal Model of Helicobacter pylori Gastritis in Humans" Gastroenterology, vol. 99, 352-361 (1990); Fox, J. G. et al. "Helicobacter felis Gastritis in Gnotobiotic Rats: An Animal Model of Helicobacter pylori Gastritis" Infect. Immun., vol. 59, 785-791 (1991); Fox, J. G. et al. "Local and Systemic Immune Responses in Murine Helicobacter felis Active Chronic Gastritis," Infect. & Immun., vol. 61, 2309-15 (1993)), have demonstrated specific IgG and IgA responses in serum and in gastric secretions in response to Helicobacter infection. However, the observation that Helicobacter pylori infection persists as a chronic infection for years, despite inducing a local and systemic immune response, is not encouraging the development of immunization strategies.
Lee et al. have reported the ability to infect germ-free rodents with Helicobacter felis, a bacterium closely related to Helicobacter pylori, and reproducibly document histologic gastritis. (Lee, A. et al. "A Small Animal Model of Human Helicobacter pylori Active Chronic Gastritis" Gastroenterology, vol. 99, 1315-1323 (1990); Fox, J. G. et al. "Helicobacter felis Gastritis in Gnotobiotic Rats: An Animal Model of Helicobacter pylori Gastritis" Infect. Immun., vol. 59, 785-791 (1991)). Since then, this bacterium-host pairing has been accepted as a good model to study Helicobacter-mediated gastritis and its initiating factors. (Lee, A. et al. "Pathogenicity of Helicobacter pylori: A Perspective" Infect. Immun., vol. 61, 1601-1610 (1993)). Infection of mice with H. felis results in a similar pathologic response to that found in humans infected with H. pylori; both types of infections result in active, chronic gastritis. (Lee et al., Gastroenterology, vol. 99, pp. 1315-1323 (1990)). Researchers have found that Helicobacter felis has the same susceptibility to antimicrobial therapy as Helicobacter pylori, and the H. felis/mouse model has been used to develop new treatments against H. pylori infection. (Dick-Hegedus, E. and Lee, A., "Use of a Mouse Model to Examine Anti-Helicobacter pylori Agents," Scand. J. Gastroenterol., vol. 26, 909-915 (1991); Chen et al., "Immunization Against Gastric Helicobacter Infection in a Mouse/Helicobacter felis Model," Lancet, vol. 339, p.1120 (1992)). Czinn et al. have shown that repetitive oral immunization with a crude lysate of Helicobacter pylori plus cholera toxin adjuvant induces a vigorous gastrointestinal IgA anti-Helicobacter pylori response in mice and ferrets. (Czinn, S. J. and Nedrud, J. G. "Oral Immunization Against Helicobacter pylori" Infect. Immun., vol. 59, 2359-2363 (1991)). In addition, Chen et al. and Czinn et al. have recently reported that oral immunization with a crude lysate of H. felis induced protection against H. felis infection in mice. (Chen, et al. "Immunization Against Gastric Helicobacter Infection in a Mouse/Helicobacter felis Model," (letter) Lancet, vol. 339,1120-1121 (1992); Czinn, S. et al. "Oral Immunization Protects Germ-Free Mice Against Infection from Helicobacter felis" Proceedings of the DDW, American Gastroenterological Association, 1321, A-331 (May 10-13, 1992); Czinn et al., Vaccine, vol. 11, 637-42 (1993)). The exact nature of the antigen(s) responsible for the induction of this protection, however, had not been determined, and no information suggested that the protective antigen(s) of H. felis that induced protection against this pathogen would induce a cross-reactive protection extending to another Helicobacter species.
We have demonstrated for the first time that Helicobacter pylori and H.felis shared antigenic determinants by obtaining monoclonal antibodies after oral immunization of mice with either Helicobacter pylori or H. felis sonicates and showing that some of these antibodies, directed against Helicobacter pylori, would crossreact with H. felis and vice versa, (Michetti, P. et al. "Specificity of Mucosal IgA Response in Balb/C Mice Following H. felis or Helicobacter pylori Challenges" Proceedings of the DDW, American Gastroenterological Association, 1001, A-251 (May 10-13, 1992); Davin, C. et al. "Helicobacter pylori Urease Elicits Protection Against H. felis Infection in Mice" Proceedings of the DDW, American Gastroenterological Association, 1213, A-304 (May 16-19, 1993)), but the basis for these cross-reactivities were unknown.
Based on the homology existing between the different known urease amino acid sequences, it has been proposed that jack bean urease could be used as a vaccine against Helicobacter pylori. (Pallen, M. J. and Clayton, C. L. "Vaccination Against Helicobacter pylori Urease" Lancet, vol. 336, 186-7 (1990)). Nevertheless, despite the homology among the different urease sequences, cross-reactivity is not the rule. Guo and Liu have shown years ago that ureases of Proteus mirabilis, Proteus vulgaris and Providencia rettqeri show cross-reactivity to each other, while ureases of jack bean and Morcanella morganii are immunologically distinct from the three former ureases. (Guo, M. and Liu, P. V. "Serological Specificities of Ureases of Proteus Species" J. Gen. Microbiol, vol. 136, 1995-2000 (1965)). So, even if an antigenic cross-reactivity of Helicobacter pylori urease with other Helicobacter ureases was a reasonable postulate, no data existed demonstrating that this was really the case until we showed that some H.filis monoclonal antibodies cross-reacted with Helicobacter pylori urease. (Davin, C. et al. "Helicobacter pylori Urease Elicits Protection Against H. felis Infection in Mice" Proceedings of the DDW, American Gastroenterological Association 1213, A-304 (May 16-19, 1993)). J. Pappo has further demonstrated that mice which have been infected by H. felis produce antibodies which crossreact with Helicobacter pylori urease but not jack bean urease (J. Pappo, unpublished data, 1993). The fact that jack bean urease does not fall in the same immunological category than Helicobacter urease suggests that jack bean urease may not be useful for immunization against Helicobacter infections, the way it was done for enteric bacteria. (Pimentel, J. L. and Cook, M. E. "Improved Growth in the Progeny of Hens Immunized with Jackbean Urease" Poultry Sci., vol. 64, 434-439 (1988)). Furthermore, attempts to immunize mice against H. felis infection by oral or intraperitoneal delivery of jack bean urease resulted in the production of antibodies against jack bean urease, but failed to protect the mice from infection. (Chen, M. et al. "Failure of Immunization Against Helicobacter Using Jack Bean Urease," Acta Gastroenterol. Belg., vol. 56, 94 (1993)).
The use of an antigen that is the reaction product of urease and glutaraldehyde is described in U.S. Pat. No. 4,837,017, "Urease Antigen Product and Process," issued Jun. 6, 1989, to LeVeen et al. The patent describes the use of the antigen to reduce ammonia toxicity caused by urea splitting organisms. LeVeen et al. disclose the injection of glutaral-dehyde treated jack bean urease into the bloodstream. The LeVeen patent does not disclose the administration of the urease antigen to the mucosal surface of a mammal in order to stimulate antibody production by the local immune system. Furthermore, there is no evidence in the specification that the injection of a jack bean urease antigen could prevent Helicobacter infection or be used to treat gastroduodenal infection by Helicobacter.
Eaton et al. have shown that mutant H. pylori cultures with weak urease activity are unable to infect gnotobiotic piglets. (Eaton et al., "Essential Role of Urease in the Pathogenesis of Gastritis Induced by Helicobacter pylori in Gnotobiotic Piglets," Gastroenterology, vol. 98, A654 (1990)). Eaton does not describe the use of a urease antigen as a vaccine to prevent Helicobacter infection or as a method of treating Helicobacter infection.
The use of Helicobacter pylori urease, or of related ureases, as a vaccine against Helicobacter pylori infection has previously been proposed by A. Labigne, and incorporated among the claims of a patent filed on Oct. 6th, 1988 by Pasteur Institute, Paris, France. (Labigne, A. "Sequences of Nucleotides Coding for a Protein Having an Urease Activity". EPO patent application # EPO 367 644 A1, 1989. International Publication # WO 90/04030, 1990). The specification of this document contains, however, no evidence of vaccination of any mammal against any Helicobacter infection with urease. This part of the Pasteur Institute patent, therefore, has not been reduced to practice, and the related claims (claims 27 and 28, page 16) should not be considered as valid. Furthermore, the claims of this document relate to a protein presenting a urease activity, and it will be understood from the experiments described below that enzymatic activity of the urease-based vaccine is not required to induce protection after oral immunization.
Moreover, while sequence homology with other bacterial ureases might support the use of urease as a vaccine candidate against Helicobacter pylori infection, the current knowledge of human Helicobacter pylori infection would certainly not. First, despite the fact that infected individuals often mount a strong antibody response to urease, the anti-urease immune response does not result in clearance or control of the infection. Second, Helicobacter pylori is able to transport urease out of the cell and to shed it from its surface, (Evans, D. J. et al. "Urease-Associated Heat Shock Protein of Helicobacter pylori" Infect. Immun., vol. 60, 2125-2127 (1992), Ferrero, R. L. and Lee, A. "The Importance of Urease in Acid Protection for the Gastric-Colonizing Bacteria Helicobacter pylori and Helicobacter felis sp. nov." Microb. Ecol. Health Dis., vol. 4, 121-134 (1991)), thus urease may not represent an appropriate target for the development of a protective mucosal immune response. Indeed, mucosal immune protection is thought to be mainly mediated by secretory IgA, the agglutinating activity of which would be impaired when the recognized antigen can be shed by the target pathogen. Third, urease appears to be toxic for epithelial cells in culture, and has been suspected to play a role in mucus degradation and in peptic ulceration in vivo (Megraud, F. et al., "Further Evidence of the Toxic Effect of Ammonia Produced by Helicobacter pylori Urease on Human Epithelial Cells," Infect. & Immun., vol. 60, 1858-63 (1992); Murakami, M. et al., "Gastric Ammonia has a Potent Ulcerogenic Action on the Rat Stomach," Gastroenterology 1993, vol. 105, 1710-15), thus its use as antigen may be toxic.
Nevertheless, we reasoned that this antigen could be a potentially efficient vaccine if:
first, we would deliver it orally at a sufficiently high dose to elicit a stronger immune response than the naturally occurring one PA1 second, the amount of antibodies produced would be high enough to bind all the urease, shed or not shed PA1 third, we would use subunits of urease or a molecular species that was non toxic.
Another aspect of the invention describes the use of antibodies directed against urease to prevent and to treat Helicobacter infection. European Patent Application No. 91310049.1, filed by Kunio Ando on Oct. 31, 1991, claiming priority on Japanese Patent Application No. 296609/90 filed Nov. 1, 1990, titled "A Method for Producing a new Medicine for Both Treating and Preventing Peptic Ulcer Diseases and Gastritis and Thus Formulated Medicines," describes the oral administration of polyclonal antibodies derived from bovine colostrum and bovine serum to patients with active chronic gastritis type B and to patients with duodenal ulcer. The Ando application describes the use of an antibody preparation directed against many antigens, including Helicobacter pylori, and does not disclose the use of an antibody directed against urease to treat or prevent Helicobacter pylori infection. The use of antibodies to treat gastric disease in gnotobiotic piglets was described in U.S. Pat. Nos. 5,258,178 and 5,260,057, issued to Cordle and Schaller and titled "Method and Product for the Treatment of Gastric Disease." The Cordle and Schaller patents describe the use of an antibody preparation that does not solely contain antibodies directed against Helicobacter pylori, and does not disclose the use of an antibody directed against urease to treat or prevent Helicobacter pylori infection. Nagata et al. describe the preparation of a monoclonal antibody directed against Helicobacter pylori that inhibits urease activity. (Nagata, K., et al., "Monoclonal Antibodies Against the Native Urease of Helicobacter pylori: Synergistic Inhibition of Urease Activity by Monoclonal Antibody Combinations," Infect. and Immun., Vol. 60,4826 (1992)). Nagata et al. do not describe the use of monoclonal antibodies directed against urease to prevent or to treat Helicobacter pylori infection.
Very few examples of therapeutic vaccines are available in the literature. Most of them are related to parenteral immunizations aimed to stimulate the host's immune system against malignant tumors, to modulate the immune system in autoimmune diseases such as rheumatoid arthritis or as desensitization in allergy states. Therapeutic vaccination procedures against different infections were also performed, most of them via a parenteral route of immunization. They included immunizations against leprosy in humans (Zaheer S A et al. "Combined Multidrug and Mycobacterium w Vaccine Therapy in Patients with Multibacillary Leprosy" J. Infect Dis., vol. 167, 401-410 (1993), Mukherjee A. et al., "Histopathological Monitoring of an Immunotherapeutic Trial with Mycobacterium w." Int. J Lepr. Other Mycobact. Dis., vol. 60, 28-35 (1992)), in complementation of antibiotic therapy, vaccination against Phythiosis insidiori, a mycological infection, in horses (Mendoza L, et al., "Evaluation of Two Vaccines for the Treatment of Pythiosis insidiosi in Horses" Mycopathologia, vol. 119, 89-95 (1992)), an uncontrolled study on the use of an autovaccine in chronic osteomyelitis (Sologub VV. "Experience in Using an Autovaccine in Treating Patients with Chronic Osteomyelitis" Vrach, Delo, 122-125 (1992)) and systemic immunization against Campylobacter fetus infection of female cattle (Schurig, G.G.D., et al., "Bovine Venereal Vibriosis: Cure of Genital Infection in Females by Systemic Immunization," Infect. & Immun., Vol. 11, 245-51 (1975)). To date, only one oral immunotherapy study aimed at stimulating the mucosal immune system in order to treat (and to prevent recurrence of) a mucosal infection has been performed, for urinary tract infection (Schulman CC, et al. "Oral Immunotherapy of Recurrent Urinary Tract Infections: A Double-Blind Placebo-Controlled Multicenter Study" J Urol., vol. 150, 917-921 (1993)). In that study, Schulman et al. used a lysate of selected E. coli strains, together with an concomitant treatment of antibiotics, chemotherapeutics or urinary tract disinfectants to treat the acute infection at entry in the study. Therefore, no study has demonstrated so far the effectiveness of a therapeutic vaccine, used as a monotherapy, administered to the mucosal immune system, against a bacterial disease.
The novelty of a therapeutic vaccine against Helicobacter infection also comes from the observation that H. pylori persists as a chronic infection in the gastric cavity for years, despite inducing a vigorous local and systemic immune response. This observation was conceptually already an obstacle to the development of a prophylactic vaccine against Helicobacter infection, but was even more an obstacle to the development of a therapeutic immunization.
In summary, there remains a need for effective treatment and prevention of Helicobacter pylori-induced gastric infection in humans. Recent data suggested the possibility to generate a vaccine against this infection, but have not provided a clear identification of defined antigen(s), common to all strains of Helicobacter pylori, that could be incorporated into a safe and effective vaccine.
In this invention, we have identified the urease antigen of Helicobacter pylori as a candidate vaccine and demonstrated its efficacy in an animal model. We have also demonstrated the use of the Helicobacter pylori urease antigen for the treatment and eradication of Helicobacter infection. We have further demonstrated that the B subunit of urease alone (ure B) is effective as a vaccine useful for the prevention of and treatment of Helicobacter infection. These results were unexpected in the light of the natural history of Helicobacter infections.